The capacity to diagnose disease is of central concern to human, animal and plant genetic studies, and particularly to inherited disease diagnostics. Genetic disease diagnosis typically is pursued by analyzing variations in DNA sequences that distinguish genomic DNA among members of a population. If such variations alter the lengths of the fragments that are generated by restriction endonuclease cleavage, the variations are referred to as restriction fragment length polymorphisms (RFLPs). Where a heritable trait is linked to a particular RFLP, the presence of the RFLP can be used to predict the likelihood that the trait will be expressed phenotypically. Statistical methods have been developed to permit the multilocus analysis of RFLPs such that complex traits that are dependent upon multiple alleles can be mapped. See S. Lander et al., 83 PROC. NAT'L ACAD. SCI. (U.S.A.) 7353-57 (1986); S. Lander et al., 84 PROC. NAT'L ACAD. SCI. (U.S.A.) 2363-67 (1986); H. Donis-Keller et al., 51 CELL 319-37 (1987); S. Lander et al., 121 GENETICS 185-99 (1989).
In some cases, DNA sequence variations are in regions of the genome that are characterized by short tandem repeats (STRs) that include tandem di- or tri-nucleotide motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. These polymorphisms are used in a large number of genetic mapping studies.
A third class of DNA sequence variations results from single nucleotide polymorphisms (SNPs), also referred to as single base polymorphisms, that exist between individuals of the same species. Such polymorphisms are far more frequent, at least in the human genome, than RFLPs or STRs and VNTRs. In some cases, such polymorphisms comprise mutations that are a determinative characteristic in a genetic disease. Indeed, such mutations may affect a single nucleotide present in a coding sequence sufficiently to cause the disease (e.g., hemophilia, sickle-cell anemia). An example of a single nucleotide polymorphism which predisposes a disease is the three-allelic polymorphism of the apolipoprotein E gene. This polymorphism is due to single base substitutions at two DNA loci on the Apo E gene (Mahley, 240 SCI. 622-30 (1988)). It may explain as much as 10% of the phenotypic variation observed in serum cholesterol levels. More that 90% of patients with type III hyperlipoproteinemia are homozygous for one of the APO E alleles.
In many cases, however, single nucleotide polymorphisms occur in non-coding regions. Single nucleotide polymorphisms in non-coding regions are often still useful as markers for predisposition to disease if a proximal relationship exists between the single nucleotide polymorphic locus and a disease-related gene. A disease-related gene is any gene that, in one or more variant is associated with, or causative of, disease. Despite the central importance of polymorphisms in modern genetics, no practical method has been developed which permits enumerative analysis of disease-associated polymorphic sites.
There is particular interest in molecular mechanisms for the diagnosis of cancer. Cancer is a disease characterized by genomic instability. The acquisition of genomic instability is thought to arise from a coincident disruption of genomic integrity and a loss of cell cycle control mechanisms. Generally, a disruption of genomic integrity is thought merely to increase the probability that a cell will engage in the multistep pathway leading to cancer. However, coupled with a loss of cell cycle control mechanisms, a disruption in genomic integrity may be sufficient to generate a population of genomically unstable neoplastic cells. A common genetic change characteristic of the early stages of transformation is a loss of heterozygosity. Loss of heterozygosity at a number of tumor suppressor genes has been implicated in tumorigenesis. For example, loss of heterozygosity at the P53 tumor suppressor locus has been correlated with various types of cancer. Ridanpaa et al., 191 PATH. RES. PRACT. 399-402 (1995). The loss of the apc and dcc tumor suppressor genes has also been associated with tumor development. Blum, 31A EUROP. J. CANCEr 1369-72 (1995).
Loss of heterozygosity is therefore a potentially useful marker for detecting the early stages of cancer. However, in the early stages of cancer only a small number of cells within a tissue have undergone transformation. Genetic changes characteristic of genomic instability theoretically can serve as markers for the early stages of, for example, colon cancer, and can be detected in DNA isolated from biopsied colonic epithelium and in some cases from transformed cells shed into fecal material. Sidransky et al., 256 SCI., 102-105 (1992).
Detection methods proposed in the art are time-consuming and expensive. Moreover, methods according to the art cannot be used to identify a loss of heterozygosity or microsatellite instability in small subpopulation of cells when the cells exist in a heterogeneous (i.e., clonally impure) sample. For example, in U.S. Pat. No. 5,527,676, it is stated that tissue samples in which a mutation is to be detected should be enriched for tumor cells in order to detect the loss of heterozygosity in a p53 gene.
The present invention provides molecular assays for the detection of nucleic acids, especially nucleic acids that are indicative of disease.